This invention relates to the fractionation of salt-containing proteinaceous solution or suspension mixtures by the total or fractional precipitation of their protein content and, more particularly, to the use of desalting techniques for inducing or enhancing such protein precipitation. Still more particularly, this invention is concerned with the use of such desalting techniques for effecting protein precipitation in combination with the addition of heavy metal ions and/or as an integral part of a selective plasmapheresis procedure for the removal or recovery of one or more selected plasma proteins from blood plasma.
It is well-known in the biochemical literature that the desalting of aqueous proteinaceous solution or suspension mixtures derived from biological fluids, i.e., the removal therefrom of the various salts and ions of alkali metals and alkaline earth metals normally present in such biological fluids, tends to cause precipitation of a protein fraction of variable and heterogeneous composition, generally known as euglobulins. A number of different techniques are well-known in the art for carrying out such desalting treatment, including, for example, passive dialysis, dilution, electrodialysis, ultrafiltration, and ion exchange chromatography. The fractionation of proteinaceous solution or suspension mixtures from biological fluids, including plasma, employing electrodialytic desalting, alone or in combination with other protein separation techniques, including forced-flow electrophoresis, electrodecantation, and alcohol precipitation, is described in the Stern U.S. Pat. No. 3,972,791, issued Aug. 3, 1976. However, this patent makes no mention whatsoever of using any desalting treatment either in combination with heavy metal ion precipitation of proteins, or as an integral part of a selective plasmapheresis procedure for the removal or recovery of one or more selected plasma proteins from blood plasma.
Heavy metal ions, such as zinc, ferrous, ferric, lead, silver, and mercury ions, are well-known in the art as exerting a precipitating action on proteinaceous materials by the reversible formation of insoluble metal-protein complexes. The use of heavy metal ions in protein precipitation and fractionation is reviewed in detail by Schultze and Heremans, "Molecular Biology of Human Proteins", Elsevier Publishing Co., New York, Vol. No. 1, pp. 259-261 (1966). The most commonly employed heavy metal ions, at least in regard to plasma protein fractionation, are zinc ions which, along with the iron-derived ions, are relatively nontoxic in comparison with the other heavy metal ions and, in fact, have beneficial nutritional effects in sufficiently limited doses. In the conventional plasma protein fractionation scheme using zinc ions for precipitating an immunoglobulin-rich fraction and leaving an immunoglobulin-impoverished albumin-rich supernatant fraction, the zinc ions are required in a concentration of 20 mM in decalcified plasma and 50 mM in citrated plasma (Pennell, R.B., in "Plasma Proteins", F.W. Putnam, Ed., Vol. I, Academic Press, 1960, p. 9). Even higher zinc ion concentrations are required for total precipitation of all proteins. Such high zinc ion concentrations in the resulting fractions must subsequently be removed, or at least significantly reduced, if such fractions are to be re-administered to human or animal recipients.
A widely used technique in the field of blood fractionation is plasmapheresis. In this technique, whole blood is withdrawn from a living donor, anticoagulated, and separated into a plasma fraction and a corpuscular element fraction, generally by centrifugation or filtration. In conventional plasmapheresis, the separated plasma fraction is retained, while the separated corpuscular element fraction is returned back into the blood stream of the donor. The primary use of plasmapheresis is the collection of plasma for subsequent preparation of purified plasma proteins employed in clinical medicine, without wasting the corpuscular elements of the donor's blood, for which there is little clinical demand. Approximately ten million plasmapheresis collections are carried out yearly in the United States for this purpose by commercial blood banks. From the plasma so-collected, only the few plasma proteins in greatest clinical demand, i.e., antihemophilia Factor VIII, immunoglobulin IgG, and serum albumin, are generally recovered, with all of the other numerous plasma proteins being mostly wasted.
In addition to its use in the preparation of clinically useful, purified plasma proteins, plasmapheresis has recently been gaining increasing attention as a modality for the direct treatment of a variety of diseases. The purpose of such therapeutic plasmapheresis is the removal from the patient's blood of pathologic plasma proteins or plasma proteins which are present in a noxiously high concentration. A variety of diseases may involve such noxious proteins. Best known among these are the gammopathies, multiple myeloma, and Waldenstrom's macroglobulinemia. The increased concentration of abnormal monoclonal immunoglobulins in the plasma of these patients may cause life-threatening hyperviscosity of the blood. Another category of diseases containing abnormal proteins are the autoimmune diseases, such as lupus erythematosus, myasthenia gravis, and, possibly, arthritis and cancer, where the primary offending components of the plasma are either specific antibodies or circulating antigen-antibody complexes. A third category of diseases may be considered as having genetically determined errors in metabolism, as for instance, homozygous familial hypercholesterolemia, characterized by abnormally high levels of circulating lipoproteins. As a treatment for these various categories of diseases, therapeutic plasmapheresis has been found to cause not only short-term palliative amelioration, but also, in certain cases, to cause long-term improvements.
In conventional plasmapheresis, whether carried out for preparative purposes or for therapeutic purposes, all of the plasma components are withdrawm from the donor's circulation, even though the main objective is the recovery or removal of not more than a few selected plasma proteins. The chief drawback of this procedure is that only a limited volume of plasma can be drawn from a given donor, if no plasma replacement is given. For more intensive treatments, the withdrawn plasma must be replaced either with purified albumin, or with normal plasma or other suitable plasma replacement fluid. This latter form of treatment is referred to as plasma exchange. Purified albumin is very expensive and does not provide all the proteins necessary for optimal replacement. Replacement with normal plasma is also expensive, and carries the risk of hepatitis. Moreover, the supply of normal plasma may soon be insufficient to fulfill the needs of all the patients who may benefit from such treatment.
In order to overcome these shortcomings of conventionaly plasmapheresis, attempts have been made to develop selective plasmapheresis techniques for selectively removing only the clinically useful or noxious plasma proteins while leaving the bulk of the remainder of the plasma components in the donor's circulation, thereby enabling extensive plasmapheresis without the need for any plasma replacement. In these selective plasmapheresis techniques, the plasma fraction, after being separated from the corpuscular element fraction, is treated so as to remove one or more selected plasma proteins therefrom, and the resulting protein-impoverished plasma fraction is thereafter recombined with the corpuscular element fraction for return back into the donor's bloodstream. In one such system, described, for example, by Terman, et al., FEBS Letters, Vol. 68, No. 1, pp. 89-94 (September, 1976), the protein-impoverished plasma fraction is obtained by passing the plasma fraction through an immunoadsorption column to cause adsorption of certain immunoglobulins and/or immune complexes. This technique offers a high degree of specificity in the profile of proteins removed, but as an on-line technique for treating plasma maintained in extracorporeal circulation during a continuous or semi-continuous flow plasmapheresis procedure, it has a rather limited potential for withdrawing large quantities of immunoglobulins from the plasma due to the limited binding capacity of the immunoadsorption column. In another selective plasmapheresis technique previously developed by the present inventor, forced-flow electrophoresis is employed for separating an immunoglobulin-rich fraction from plasma on the basis of differences in electrophoretic mobility. Bier, et al., Trans. Amer. Soc. Artif. Int. Organs, Vol. 16, pp. 325-333 (1970). While this technique offers the potential of withdrawing large quantities of immunoglobulins and the additional advantage of yielding them in a readily accessible form, forced-flow electrophoresis is rather time-consuming and cumbersome to implement as an on-line technique for treating plasma maintained in extracorporeal circulation during a continous or semi-continuous flow plasmapheresis procedure. It should be noted that neither one of these two previously proposed selective plasmapheresis techniques involves the separation of proteins based upon their differences in solubility.